Bioplastics
Bioplastics are biologically produced polymers with many of the properties of petroleum-derived plastics. Bioplastics are long carbon and oxygen chain polyesters, the basic chemical structure of which is: ##STR1## The carbon to oxygen linkage is in the ester form. If the R group of the structure described above is an alkyl group, the structure is a polyhydroxyalkanoate (PHA). Most typically, R is a carbon chain of between 1 and 10 residues. R groups such as methyl, ethyl, propyl and butyl have been found in bioplastics.
Poly-beta-hydroxybutyric acid (PHB) is a form of PHA found as an intracellular storage compound in many species of bacteria. PHB was identified in Bacillus megaterium in 1925. (Lemoigne, M. Ann Inst. Pasteur. Paris., 35: 144, 1925). Referring to the structure described above, if R is a methyl group, the compound is PHB. PHB is a biodegradable thermoplastic that serves as a carbon and energy source for the bacterium. Due to its high degree of crystallinity, PHB is hard and brittle. Holmes, et al., U.S. Pat. No. 4,393,167, discusses the use of PHB and PHB blends.
In many bacteria, PHB is synthesized via a three-step metabolic pathway in which the enzymes ketothiolase, NADP-dependent acetoactyl-CoA reductase, and PHB synthase (PHB polymerase) catalyze the conversion of acetyl CoA to PHB (Dawes and Senior, Adv. Microb. Physiol. 10: 135-266, 1973). The genes corresponding to these three enzymes have been cloned from Alcaligenes eutrophus. E. coli can be made to synthesize PHB after transformation with these genes (Slater et al., J. Bacteriol. 170: 4431-4436, 1988; Schubert et al., J. Bacteriol. 170: 5837-5847, 1988; Slater et al., Applied and Environmental Microbiology, 58: 1085-1094, 1992; Peoples and Sinskey, J. Biol. Chem., 264: 15298-15303, 1989; Peoples and Sinskey, J. Biol. Chem., 264: 15293-15297, 1989).
Bacterial genera producing bioplastics include Alcaligenes, Athiorhodium, Azotobacter, Bacillus, norcardia, Pseudomonas, Rhizobium, Spirillium, Zoogloea and Rhodococcus (Haywood et al. Biotech. Lett. 11: 471-476, 1989). Depending on the nutrient source, the bacteria incorporate hetropolymers of the D-isomer of the beta-hydroxyalkanoates. (Brandl et al., Int. J. Bio. Macromol., 11: 49-55, 1989; Gross et al. Macromolecule, 22: 1106-1115, 1989). Thus, polymers containing 3-hydroxybutyrate units (3HB), 3-hydroxyvalerate units (3HV), 3-hydroxypropionate (3HP) units and 5-hydroxyvalerate (5HV) units have been produced under controlled conditions (European Patent Application 0 440 165 A2).
Due to its high degree of crystallinity, PHB is hard and brittle. On the other hand, copolymers of 3HB, 3HV, 3HP and 5HV may have a number of advantages in terms of moldability, thermal resistance to degradation, or impact resistance. By using specific carbon sources one may be able to incorporate unusual repeating units such as branched alkyl, bromo or phenyl groups in the molecule. (Lenz et al. in Novel Biodegradable Microbial Polymers. Ed: Dawes, E. A., vol. 186, pp. 23-25, 1990). Thus, it is very likely that microorganisms have the ability to incorporate various monomers other than D(-)-hydroxybutyrate into the polymer chain. Characteristics of PHB synthase enzyme determines the type of polymers synthesized. A. eutrophus can accumulate PHA containing C.sub.4 and C.sub.5 units while P. Oleovarans forms a PHA containing C.sub.8 units. Rodospirillum rubrum produce PHA of C.sub.4 to C.sub.7 units. P. putida, P. oleovarans, P. aeruginosa, P. flurescens and P. testeronii were able to accumulate PHAs containing 3-hydroxyacid units in the range of C.sub.5 to C.sub.10. (Haywood et al., Biotech. Lett. 11: 471-476, 1989). In this regard, identification of different PHB synthase genes and their characterization in in vitro systems will permit the production of various novel polymers. Such substrates include C.sub.5 -C.sub.8 linear 3-oxo thiolesters, oxoesters and methylene ketones (Peoples and Sinskey, WO 91/00917).
Bioplastics have properties that are advantageous for the plastics industry. Unlike synthetic plastics, bioplastics are biodegradable and could eventually become a renewable source of plastic that is not dependent on petroleum. PHAs can be flexible and moldable. Additionally, bioplastics are biocompatible. Because of these properties, bioplastics can advantageously be used in place of synthetic plastics.
Expression of bioplastics in plants such as corn and potatoes has been suggested. See WO 91 00917; Pool, Science 245: 1187-9, 1989. PHB has been expressed in recombinant Arabidopsis thaliana plants. Poirer et al., Science, 256: 520-523 (1992).
Genetic Engineering of Cotton
Although successful transformation and regeneration techniques have been demonstrated in model plants species such as tobacco (Barton et al., Cell 32: 1033-1043, 1983), similar results with cotton have only been achieved relatively recently. See, e.g. Umbeck et al. Bio/Technology, 5[3] 263-266 (1987); Firoozabady et al., Plant Mol. Bio. 10: 105-116 (1987); Finer and McMullen., Plant Cell Rep. 8: 586-589, 1990.
Cotton is one of the most important cash crops. Successful transformation and regeneration of genetically engineered cotton plants has the potential to be of significant value to this agriculturally important crop. One of the most important benefits potentially achievable from genetically engineering cotton plants is the alteration and modification of cotton fiber quantity and quality.
Cotton fiber (seed hair) is a differentiated single epidermal cell of the ovule. At maturity the fiber cell consists of a cell lumen, primary cell-wall and secondary cell-wall. The primary cell-wall is made up of pectic compounds, cellulose, and small amounts of protein. The secondary cell-wall consists of cellulose. At maturity, the cotton fiber contains 87% cellulose.
Cotton fiber development can be divided into initiation, primary cell-wall synthesis stage, secondary cell-wall deposition stage, and maturation phases. Many hundreds of genes are required for the differentiation and development of cotton fiber. Work on in vitro translated fiber proteins (Delmar et al., J. Cell Sci. 2: 33-50, 1985) and protein isolated from fiber (Graves and Stewart, J. Exp. Bot. 39: 59-69, 1988) clearly suggests differential gene expression during various developmental stages of the cell. Only a few of the genes involved in the biosynthesis of the large numbers of fiber-specific structural proteins, enzymes, polysaccharides, waxes or lignins have been identified (John and Crow, Proc. Natl. Acad. Sci. USA, 89: 5769-5773, 1992). Since these genes and their interactions with environment determine the quality of fiber, their identification and characterization is considered to be an important aspect of cotton crop improvement.
The quality of the cotton fiber is dependent on such factors as the extent of elongation and degree of secondary wall deposition. It is assumed that both a number of genes and environmental factors regulate the physical characteristics of the fiber such as length, strength and micronaire value. However, the genes responsible for cellulose synthesis and fiber development in cotton plants are heretofore entirely uncharacterized at a molecular level.
The most commercially useful plant fiber is derived from cotton (Gossypium arboreum, Gossypium herbaceum, Gossypium barbadense and Gossypium hirsutum). However, there are other fiber-producing plants with a potential commercial use. These plants include the silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax.
Promoters
Promoters are DNA elements that direct the transcription of RNA in cells. Together with other regulatory elements that specify tissue and temporal specificity of gene expression, promoters control the development of organisms. Thus, there has been a concerted effort in identifying and isolating promoters from a wide variety of plants and animals.
Many promoters function properly in heterologous systems. For example, promoters taken from plant genes such as rbcS, Cab, chalcone synthase and protease inhibitor from tobacco and Arabidopsis are functional in heterologous transgenic plants. (Reviewed by Benfey and Chua, Science 244: 174-181, 1989). Specific examples of transgenic plants include tissue-specific and developmentally regulated expression of soybean 7s seed storage protein gene in transgenic tobacco plants (Chen, et al. EMBO J. 7: 297-302, 1988.) and light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene promoter in transgenic tobacco (Ha and An, Proc. Natl. Acad. Sci. USA 85: 8017-8021, 1988). Similarly, anaerobically inducible maize sucrose synthase-1 promoter activity was demonstrated in transgenic tobacco (Yang and Russell, Proc. Natl. Acad. Sci USA, 87: 4144-4148, 1990). Tomato pollen promoters were found to direct tissue-specific and developmentally regulated gene expression in transgenic Arabidopsis and tobacco (Twell et al., Development 109: 705-713, 1990). Similarly, one cotton promoter has been shown to express a transgene in a fiber-specific manner (John and Crow, Proc. Natl. Acad. Sci. USA, 89: 5769-5773, 1992). Thus, some plant promoters can be utilized to express foreign proteins in specific tissues in a developmentally regulated fashion.
Many of the features of bioplastics could be advantageously combined with plant fiber. However, bioplastic-containing fiber-producing plants have neither been proposed nor created. What is needed in the art of molecular biology is a cotton plant containing heterologous bioplastic.